Oligodeoxynucleotide intervention for prevention and treatment of sepsis

ABSTRACT

A method for producing ODNs in bacterial or fungal cells in vivo for treatment of sepsis so that, when the ODNs reach and knock down their target genes, and thereby kill bacterial or fungal cells or inhibit their growth, the bacterial or fungal accumulation in the bloodstream is held constant or diminished and the sepsis syndrome is reduced or eliminated. The invention also contemplates of certain ODNs for use in treatment of sepsis.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part of co-pendingapplication Ser. No. 10/453,410, filed Jun. 3, 2003 and IDENTIFICATIONOF NOVEL ANTIBACTERIAL AGENTS BY SCREENING THE SINGLE-STRANDED DNAEXPRESSION LIBRARY, and co-pending application Ser. No. 10/743,956,filed Dec. 23, 2003 and entitled OLIGODOXYNUCLEOTIDE (ODN) LIBRARIES,THEIR USE IN SCREENING FOR ANTIBACTERIAL AGENTS, AND CATALYTIC ODNSEQUENCE FOR USE AS AN ANTIBACTERIAL AGENT. Both of the applicationslisted in this paragraph are hereby incorporated into the specificationof the present application in their entirety by this specific referencethereto.

BACKGROUND OF THE INVENTION

Severe sepsis and septic shock are life threatening complication ofinfections and the most common cause of death in intensive care units(Angus et al., 2001, Crit. Care Med., 29:1303-1310). Recent US andEuropean surveys have estimated that severe sepsis accounts for 2-11% ofall admissions to hospital or intensive care units (Martin et al., 2003,New Engl. J. Med., 348, 1546-1554). The incidence of this condition isrising due to the aging of the population and increasing numbers ofimmuno-compromised and critically ill patients.

Sepsis comprises a complex clinical syndrome that results from thebody's response to infection caused by bacterial and/or fungal pathogensinvading the body (Cohen, 2002, Nature, 420, 885-891). Normally, apotent, complex, immunologic cascade ensures a prompt, protectiveresponse to microorganism invasion in humans. However, a deficientimmunologic defense may allow infection to become established. Further,an excessive or poorly regulated response may harm the host throughmaladaptive release of indigenously generated inflammatory compounds.Additionally, diabetic individuals or others who suffer from lymphedema,especially in the feet and legs, are at risk of infection from exogenousopportunistic pathogens normally present on the skin due to the growthpotential afforded by the edematous medium or the lack of circulation.Such infections may result in severe cellulitis or related sequelae thatcan lead to sepsis.

Gram negative bacilli (mainly Escherichia coli, Klebsiella species, andPseudomonas aeruginosa) and Gram positive cocci (mainly staphylococciand streptococci) are the most common microbial pathogens isolated frompatients with severe sepsis and septic shock. (Bochud, 2001, IntensiveCare Med., 27 (Suppl 1): S33-S48). Although infections by Gram negativebacteria were predominant in the 1960s and early 1970s, Gram positiveinfections have increased in the past two decades and now account forabout half of cases of severe sepsis. Fungi, mostly Candida, account forabout 5% of all cases of severe sepsis, but these fungal infections arealso increasing in many countries.

Gram negative infections usually occur in the lung, abdomen,bloodstream, or urinary tract. Endotoxins in the form oflipopolysaccharides (LPS), are an important component of the outermembrane of Gram negative bacteria, and have a pivotal role in inducingGram negative sepsis (Alexander & Rietschel, 2001, J. Endotoxin Res.,7:167-202). LPS binding protein in host cells binds to LPS in thebacteria and transfers it to CD14 (Ulevitch & Tobias, 1999, Curr. Opin.Immunol., 11:19-22), a protein anchored in the outer leaflet of theplasma membrane. A series of remarkable investigations have recently ledto the identification of a Toll-like receptor 4 (TLR4) as theco-receptor for LPS (Ulevitch & Tobias, 1999, Curr. Opin. Immunol.,11:19-22).

Gram positive bacteria are usually responsible for infections of skinand soft tissue, infections associated with intravascular devices,primary bloodstream infections, or respiratory infections. It isgenerally thought that the distinct cell wall substances ofgram-positive bacteria and fungi trigger a similar cascade of events asgram negative bacteria, although the structures involved are notgenerally as well studied as gram-negative endotoxin. Gram positivebacteria can cause sepsis by at least two mechanisms: by producingexotoxins that act as superantigens and by components of their cellwalls stimulating immune cells (Calandra, 2001, J. Chemother.,13:173-180).

Superantigens are molecules that bind to MHC class II molecules ofantigen presenting cells and to V^(β) chains of T cell receptors. Indoing so, they activate large numbers of T cells to produce massiveamounts of proinflammatory cytokines. Staphylococcal enterotoxins, toxicshock syndrome toxin-1, and streptococcal pyrogenic exotoxins areexamples of bacterial superantigens.

Gram positive bacteria without exotoxins can also induce shock, probablyby stimulating innate immune responses through similar mechanisms tothose in Gram negative sepsis. Indeed, Toll-like receptor 2 (TLR2) hasbeen shown to mediate cellular responses to heat killed Gram positivebacteria and their cell wall structures (peptidoglycan, lipoproteins,lipoteichoic acid, and phenol soluble modulin) (Takeuchi et al., 1999,Immunity, 11:443-451).

Following the initial host-microbial interaction, there is widespreadactivation of the innate immune response, releasing the classicpro-inflammatory cytokines IL-1, IL-6 TNF-α, and many other cytokinesincluding IL-12, IL-15, and IL18. This, in turn, activates a secondlevel of inflammatory cascades including cytokines, lipid mediators andreactive oxygen species. These early immune responses have directdamaging actions on the vascular endothelium (Wheeler et al, 1999, N.Engl. J. Med., 340:207-214). The endothelial damage causes furtherexacerbation of inflammation, resulting in neutrophil activation,neutrophil-endothelial cell adhesion, and further elaboration ofinflammatory cytokines (Esmon, 1998, Immunologist, 6, 84-89). Theseinflammatory processes further contribute to vascular endothelialdysfunction. Concurrently, the endothelial cells release Tissue Factor(TF), triggering the extrinsic coagulation cascade and accelerating theproduction of thrombin (Carvalho & Freeman, J. Crit. Illness, 9, 51-75;Esmon, 1998, Immunologist, 6, 84-89). This uncontrolled cascade ofinflammation and coagulation fuels the progression of sepsis, resultingin hypoxia, widespread ischemia, organ dysfunction, and ultimately deathfor a large number of patients.

Numerous adjunctive treatments (that is, other than antibiotics andsupportive care) for severe sepsis and septic shock have been tested inclinical trials. These include neutralization of microbial toxins suchas LPS, non-specific anti-inflammatory and immunosuppressive drugs,neutralization of pro-inflammatory cytokines, and correction ofabnormalities in coagulation. The results have been mixed (Vincent etal., 2002, Clin. Infect. Dis., 34:1084-1093), and it does not appearthat any one treatment addresses all sepsis conditions and/or causativeagents.

As recited above, the lipo-polysaccharide endotoxin found in the cellwall of gram-negative bacteria plays a key role in initiating thehumoral cascades observed in septic shock. Several anti-endotoxinantibody products have been developed and have undergone human trials(Ziegler et al., 1982, N. Engl. J. Med., 307, 1225-1230; Angus et al.,2000, JAMA, 283, 1723-1730; McCloskey et al., 1994, Ann. Intern. Med.,121, 1-5). However, despite some encouraging results from early studies,none of the anti-endotoxin strategies have been shown to be of benefitin large clinical trials.

Serum levels of tumor necrosis factor (TNF) and inter-leukin-1 (IL-1)are elevated in patients with septic shock. Both produce hemodynamiceffects that duplicate those found in sepsis, and studies indicate thatboth mediators play key roles in sepsis and septic shock such that TNFmay be a central mediator in sepsis. Similar to anti-endotoxinantibodies, antibodies to TNF or IL-1 are hypothesized to be useful inseptic shock. However, anti-TNF or anti-IL-1 antibodies have yet to bedemonstrated any beneficial effect in sepsis or septic shock (Abraham etal., 1998, Lancet, 351, 929-933).

While theoretical and experimental animal evidence exists supporting theuse of large doses of corticosteroids in severe sepsis and septic shock,all randomized human studies found that corticosteroids do not preventthe development of septic shock, reverse the shock state, or improvemortality. Lefering & Neugebauer, 1995, Crit. Care Med., 23, 1294-1303;Cronin, 1995, Crit. Care Med., 23, 1430-1439.

Coagulation abnormalities, especially disseminated intravascularcoagulation, are common in patients with sepsis and microvascularthrombosis. The ensuing tissue damage may have an important role in thepathophysiology of organ dysfunction. Treatment with activated proteinC, a protein with anti-thrombotic, pro-fibrinolytic, andanti-inflammatory effects, reduces mortality from severe sepsis (Bernardet al., 2001, N. Engl. J. Med., 344: 699-709). So far as is known, inthe current market, it is the only drug to treat sepsis. However,treatment with this drug results in relatively modest improvements inpatient mortality, and at the price of a slight increase in bleedingevents (Id.).

Prophylactic administration of antibiotics is still the main treatmentof choice for sepsis in hospital (Cunha, 1995, Med. Clin. North Am., 79,551-558). Antibiotics must be broad spectrum and cover gram-positive,gram-negative, and anaerobic bacteria because all classes of theseorganisms produce identical clinical presentations. Antibiotics must beadministered parenterally in doses adequate to achieve bactericidalserum levels. Many studies have found that clinical improvementcorrelates with the achievement of serum bactericidal levels rather thanthe number of antibiotics administered.

Unfortunately, numerous classes of antibiotics have become lesseffective as a result of the rapid emergence of antibiotic resistance bymany common bacterial pathogens such as S. aureus, Streptococcuspneumoniae and Enterococcus faecalis (Nicolaou & Boddy, 2001, ScientificAmerican, p.56-61). Methicillin-resistant S. aureus (MRSA),penicillin-resistant S. pneumococcus and vancomycin-resistant E.faecalis (VRE) are now common pathogens that are difficult to treateffectively (Pfaller, et al., 1998, Antimicrobial Agents andChemotherapy, 42:1762-1770; Jones, et al., 1999, Microbiol. Infect.Dis., 33:101-112). Probably more alarming is the emergence of multi-drugresistance pathogens (Swartz, 1994, Proc Natl. Acad. Sci. USA,91:2420-2427; Baquero, 1997, J. Antimicrobial Chemotherapy, 39:1-6).Opportunistic fungal pathogens resistant to antifungal agents have alsobeen increasingly documented in recent years and their frequency willlikely continue to increase (Rex, 1997, Clin. Infect. Dis., 24:235-247).Candida spp., Cryptococcus neoformans, and Aspergillus spp. are amongthe leading fungi responsible for the invasive infections, andantifungal resistance has been described with each of these fungi.

Until recently, the principal approach of the pharmaceutical industry tothis growing problem has been to seek incremental improvements inexisting drugs (Piddock, 1998, Curr. Opin. Microbiol., 1, 502-508).Although these approaches make a significant contribution to fightingagainst bacterial infections, difficulty remains in meeting theincreasing needs of the medical community. Thus, there is an urgent needfor new discovery strategies to discover and develop new classes ofantibiotics.

Recent advances in DNA sequencing technology have made it possible toelucidate the entire genome sequences of pathogenic bacteria and fungi.Such sequence information provides the necessary information to identifypotential gene targets, and therefore enable construction ofoligodeoxynucleotides (ODNs) for anti-bacterial or anti-fungal use.Oligonucleotide-mediated intervention (OMI) technology provides apowerful set of tools to alter the activity of any gene of knownsequence at the genomic level, including triplex formingoligonucleotides for targeted gene expression, at the messenger RNA(mRNA) level using antisense, competitively inhibitory and DNA enzymeoligos and at the protein level using ssDNA as aptamers (Chen, 2002,Expert Opin. Biol. Ther. 2(7) 735-740). This technology has shownpotential for developing highly specific and efficacious antibacterialagents (Harth et al., 2000, Proc. Natl. Acad. Sci. U.S.A, 97, 418-423;Good & Nielsen, 1998, Nature Biotech., 16, 355-358; Gasparro et al.,1991, Antisense Research and Development, 1, 117-140). Two patentssuggesting a role for AS-ODN molecules in the regulation of bacterialgrowth have been issued (McDevitt et al., 2002, J. Appl. Microbiol.Symp. Suppl., 92, 28s-34s).

Antisense, DNA enzyme, triplex, competitive inhibition and aptamertechnologies provide an efficient alternative to more difficult methodssuch as creating gene knockout in cells and organisms. Antisenseoligonucleotides (ODNs) block gene expression by Watson-Crick basepairing between an ODN and its target mRNA thereby preventingtranslation of that MRNA by Ribosomes. (Crooke, 1999, Biochim. Biophys.Acta 1489:31-44). Antisense ODNs have been used to inhibit geneexpression in eukaryotic cells and have been used to validate genetargets, and there is one antisense ODN-based product in the market anda number of others in advanced clinical trials (Uhlman, 2001, ExpertOpinion on Biological Therapy, 1:319-328). However, antisense technologyis not used extensively in prokaryotic systems. Prokaryotic cells (thecells involved in sepsis) have themselves developed endogenous antisensemechanisms for gene regulation (Simons & Kleckner, 1988, Annu. Rev.Genet., 22:567-600). Earlier results indicated that gene expression inbacteria may be accessible to inhibition by modified ODNs (Jayayaramanet al., 1981, Proc. Natl. Acad. Sci. USA, 78:1537-1541; Gasparro et al.,1991, Antisense Res Dev., 1:117-140) and that peptide nucleic acid (PNA)can inhibit gene expression in bacteria (Good & Nielsen, 1998, NatureBiotech., 16:355-358). PNA, a DNA mimic in which the nucleotide basesare attached to a pseudo-peptide backbone, hybridizes with complementaryDNA, RNA, or PNA oligomers through Watson-Crick base pairing and helixformation.

Techniques using a screening library such as is described in co-pendingU.S. pat. application Ser. No. 10/453,410, assigned to the same Assigneeas the present application, have enabled both identification of genescritical to bacterial viability and the ODNs effective in silencingthose critical genes, thus inhibiting bacterial growth/replication orkilling the bacteria. The application of these ODNs and their expressionplasmids to control bacterial and/or fungal pathogens causing sepsis isone focus of the present invention. On the other hand, sepsis representsan excessive innate immune response to microbial products, and manyprocesses in the complex pathophysiology of sepsis are simultaneouslyover-activated. The complexity of this response affords ample targetsfor ODN therapy. For that reason, the design and application of ODNs andtheir expression plasmids to down-regulate the over-activated processesis another focus of the present invention. It is, therefore, an objectof the present invention to identify the genes necessary for bacterialand fungal viability, and the host genes associated with the exaggeratedinnate immune response in sepsis.

An additional object of the present invention is to provide ODNs, andtheir sequences, that will knock down (silence) the bacterial, fungal,and host genes above.

An additional object of the present invention is to provide the saidODNs, and their sequences, as therapeutic anti-sepsis agents.

An additional object of the present invention is to identify deliverymeans of the said transfecting therapeutic ODN into target bacterial orfungal cells.

An additional object of the present invention is to provide a method ofthe treatment of bacterial or fungal sepsis using the said ODNs.

An additional object of the present invention is to provide plasmidconstructions that are used to knock down the bacterial, fungal, andhost genes above.

An additional object of the present invention is to provide the plasmidconstructions that are used as therapeutic anti-sepsis agents.

An additional object of the present invention is to identify deliverymeans of the transfecting therapeutic ODN into target bacterial orfungal cells.

Still another object of the present invention is to provide a method oftreatment of sepsis in an animal patient comprising the steps ofcontacting the causative agent of sepsis with an ODN comprising asequence targeted to a specific gene of the causative agent for alteringthe expression of the specific gene to inhibit growth of the causativeagent, kill the causative agent, or inhibit the synthesis or secretionof toxin by the causative agent.

An additional object of the present invention is to provide a method ofthe treatment of bacterial or fungal sepsis using a therapeutic ODN.

SUMMARY OF THE INVENTION

The present invention relates to a new strategy for combating sepsis.The present invention comprises a list of genes critical to bacterialand fungal viability, a list of gene associated with the exaggeratedinnate immune response in sepsis, a list of ODNs (and their DNAsequences) effective in knocking down the said genes, a list of the saidODNs (and their DNA sequences) as therapeutic anti-sepsis agents, amethod of using the said ODNs to treat bacterial sepsis, a list ofplasmid constructions effective in knocking down said genes, a list ofsaid plasmid constructions as therapeutic anti-sepsis agents, a deliverymethod of said plasmid constructions into bacterial cells, a deliverymethod of said plasmid construction into fungal cells, a delivery methodof said plasmid constructions into host cells, a method of using saidplasmid constructions to treat sepsis.

The present invention comprises methods for producing ODNs in bacterialor fungal cells in vivo so that, when said ODNs reach and knock downtheir target genes, and thereby kill bacterial or fungal cells orinhibit their growth, the bacterial or fungal accumulation in thebloodstream would be held constant or diminished and the sepsis syndromewould be reduced or eliminated.

The present invention comprises a method for producing ODNs in hostcells in vivo so that, when the said ODNs silence their target genes,the host exaggerated innate immune response would be abrogated andthereby, the sepsis syndrome would be relieved.

The present invention comprises a method for delivering ODN-expressingplasmid constructions into bacterial or fungal cells in vivo so that,when the ODNs are produced intracellularly and knock down their targetgenes, and thereby kill bacterial or fungal cells, the bacterial orfungal accumulation in the bloodstream would be diminished and thereby,the sepsis syndrome would be relieved.

The present invention comprises a method for delivering theODN-expressing plasmid constructions into host cells in vivo so that,when the ODNs are produced intracellularly and knock down their targetgenes, the host exaggerated innate immune response would be abrogatedand thereby, the sepsis syndrome would be relieved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows survival of infected mice with or without ODN treatment.This log-phase preparation of bacteria was diluted in PBS, and 3×10e8CFU of bacteria was i.p. injected to induce mouse sepsis. To treat micewith ODN, the ODN was either mixed with bacteria in vitro and then wasinjected to mice, or mice were pretreated by ODN before infection. Serumwas gathered and proinflammatory cytokines (IL-6, TNF, IL-1) andbacterial load tested, and mouse behavior monitored at various timepoints after injection.

FIG. 2 shows changes in mouse proinflammatory cytokine IL-6. Serum wascollected at 4 hr and 24 hr after bacterial infection, and IL-6concentration was measured using commercial kit.

FIG. 3 shows bacterial growth inhibition by ODN. Immediately afterdiluting the O/N cultures 1/50, ODN was added to final concentration of4 uM, 40 μM or 400 μM, with addition of equal volume water as a negativecontrol, and incubated with shaking at 30° C. After 2, 4 or 6 h, thegrowth was measured viable cell count, which was done by diluting thecultures and plating them on LB plates with streptomycin.

FIG. 4 shows bacterial growth inhibition by ODN expression plasmidAS830103. The competent cells XL10-gold(kan) were transformed with theODN expression plasmid or the plasmid without ODN insert, and platedonto LB media with chloramphenicol and incubated at 37° C. O/N.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a new strategy for combating sepsiscaused by bacterial and fungal pathogens, wherein selected ODNs and theexpression plasmid used to produce them were used as therapeuticanti-sepsis agents.

Examples of sepsis that can be treated in accordance with the presentinvention include, but are not limited to, those caused by infections inthe lung, abdomen, bloodstream, skin, soft tissue, caused by infectionsassociated with intravascular devices, or caused by respiratoryinfections.

Examples of microorganisms that can be treated in accordance with thepresent invention include, but are not limited to, Gram-negativebacteria such as Bacteroides, Fusobacterium, Escherichia, Klebsiella,Salmonella, Shigella, Proteus, Pseudomonas, Vibrio, Legionella,Haemophilus, Bordetella, Brucella, Campylobacter, Neisseria,Branhamella; Gram-positive bacteria such as Streptococcus,Staphylococcus, Peptococcus, Bacillus, Listeria, Clostridium,Propionebacteria; organisms that stain poorly or not at all with Gram'sstain such as Mycobacteria, Treponema, Leptospira, Borrelia, Mycoplasma,Clamydia, Rickettsia and Coxiella; and Fungi such as Candida,Aspergillosis, Blastomycosis, Coccidioidomycosis, Cryptococcosis,Histoplasmosis, Paracoccidiomycosis, Sporotrichosis, Zygomycosis.

Examples of bacterial target genes that can be knocked down inaccordance with the present invention include, but are not limited to,those identified from library screening and those chosen based uponknowledge about bacterial physiology. A target gene can be found amongthose involved in one of the major process complexes: cell division,cell wall synthesis, protein synthesis (translation), nucleic acidsynthesis, fatty acid metabolism, and gene regulation. Therefore,examples of bacterial target genes that can be knocked down inaccordance with the present invention include, but are not limited toFtsZ, MurB, acpP, 16s rRNA, PBPs, DNAA, DNAC, pcrA, rpoB, rpoA, rpoC,rpsC, rpsD, rpsF, rpsi, rpsJ, rpsM, rpsR, FabK, FabH, rplB, rplC, rplJ,rplK, rplM, rplN, rplO, rplP, rplR, rplT, rplV, rplX, rpmA, rpmL, valS,serS, proS, cysS, alaS, pheS, sporC, tsf, tufA, fus, secA, secV, pyrc.

The target genes critical to fungal viability can be found among thoseinvolved in one of the major process complexes: cell division, cell wallsynthesis, protein synthesis (translation), nucleic acid synthesis,fatty acid metabolism, and gene regulation. Therefore, examples offungal target genes that can be knocked down in accordance with thepresent invention include, but are not limited to, ERG1, ERG2, ERG3,ERG4, FRG5, ERG6, ERG7, ERGI11, ERG24, ERG25, ERGX, ERGY, CHS1, CHS2,CHS3, CWP1, CWP2, KRE1, KRE2, KRE5, KRE11, TIP1, GFA1.

ODN technology can down-regulate the over-expression of the host genesassociated with the exaggerates innate immune response in sepsis, sothat appropriate host response to an infection remains. Examples of hosttarget genes that can be knocked down in accordance with the presentinvention include, but are not limited to, tumor necrosis factor (TNF),interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12),interleukin-15 (IL15), nitric oxide synthase (NOS), high mobility group1 protein (HMG-1), migration inhibitory factor (MIF), Kinins,platelet-activating factor receptor antagonist (PAFra), solublephospholipase A2 (sPLA2). In particular, TNF is considered to be one ofthe most important inflammatory mediators in the sepsis cascade. TNF issignificantly elevated during sepsis (Casey et al., 1993, Ann. Intern.Med. 119:771-778; van der Poll and Lowry, 1995, Shock, 1-12), and levelsof TNF have been associated with severity of sepsis and clinical outcome(Calandra et al., 1990, J. Infect. Dis. 161:982-987; Cannon et al.,1990, J. Infect. Dis., 161:79-84). Moreover, most of the deleteriouseffects of sepsis can be mimicked by the administration of TNF (Okusawaet al., 1988, J. Clin. Invest., 81:1162-1172; Natanson et al., 1989, J.Exp. Med., 169:823-832).

Examples of the ODN therapeutics that are used to treat sepsis inaccordance with the present invention include, but are not limited to,CYXO080103, wherein, its sequence is 5′(CTT TCA ACA GTT TTG ATG ACC TTTGCT GAC CAT ACA ATT GCG ATA TCG TGG GGA GTG AGA G)3′, and its potentialtargets are btuE (GenBank ID: NP_(—)416225.1), CaiB (GenBank ID:NP_(—)414580.1), ydgD (GenBank ID: NP_(—)418152.1), ygcQ (GenBank ID:NP_(—)417249.2), ftsH (GenBank ID: NP_(—)417645.1), ppiB (GenBank ID:NP_(—)415058.1), yihl (GenBank ID: NP_(—)418308.1), zntA (GenBank ID:NP_(—)417926.1), yicI (GenBank ID: NP_(—)418116.1), fhuA(GenBank ID:NP_(—)414692.1), rpID (GenBank ID: NP_(—)417778.1), ilvB (GenBank ID:NP_(—)418127.1), lepB (GenBank ID: NP_(—)417063.1), aroK (GenBank ID:NP_(—)417849.1), mfd (GenBank ID: NP_(—)415632.1), ripA (GenBank ID:NP_(—)415166.1), accA (GenBank ID: NP_(—)414727.1), pgpA (GenBank ID:NP_(—)414952.1); CYGXacpP, wherein, its sequence is 5′(CTC ATA CTC T)3′in PNA form, and its target is the bacterial essential fatty acidbiosynthesis gene acpP (DenBank ID: NP_(—)309499); CYGXFtsZDZ. wherein,its sequence is 5′(GTT TCG AAG GCT AGC TAC AAC GAT CAT CCA G)3′, and itstarget is the bacterial essential cell division gene FtsZ (GenBank ID:NP_(—)308126).

Examples of the DNA therapeutics that are used to treat sepsis inaccordance with the present invention include, but are not limited to,regular ODN and its expression plasmid, its modification forms such aslocked nucleic acids (LNA), peptide nucleic acids (PNA),phosphorothioates, or phosphorothioates morpholino oligomer (PMO).

Examples of means to deliver the said ODN and the said ODN expressionplasmids into bacterial or fungal cells for treatment of sepsis, inaccordance with the present invention include, but are not limited to,cationic polymers such as PEI, EPEI, and porphyrins; peptides such asLys Phe Phe Lys Phe Phe Lys Phe Phe Lys, Xaa Xaa Xaa Lys Lys Arg Arg XaaXaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp Trp Xaa Xaa Xaa, Lys Xaa XaaTrp Trp Glu Thr Trp Trp Xaa Xaa Ser Gln Pro Lys Lys Xaa Arg Lys Xaa, TyrGly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp Trp Glu Thr Trp Trp Thr GluXaa, wherein any Xaa can be any amino acid. The said ODN expressionplasmids can also be delivered into bacterial cells by packaging thesaid plasmids into infectious particles using phage extracts, asdetailed below in Example 6.

Examples of means to deliver the said ODN and the said ODN expressionplasmids into host cells for treatment of sepsis, in accordance with thepresent invention include, but are not limited to, direct injection ofnaked DNA; cationic polymers such as PEI, EPEI, and porphyrins; peptidessuch as Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys, Xaa Xaa Xaa Lys Lys ArgArg Xaa Xaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp Trp Xaa Xaa Xaa, LysXaa Xaa Trp Trp Glu Thr Trp Trp Xaa Xaa Ser Gln Pro Lys Lys Xaa Arg LysXaa, Tyr Gly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp Trp Glu Thr Trp TrpThr Glu Xaa, wherein any Xaa can be any amino acid. Examples of means todeliver the said ODN expression plasmids into host cells for treatmentof sepsis, in accordance with the present invention include, but are notlimited to, viral vectors such as retroviruses and adenoviruses; directinjection of naked DNA; cationic liposome such as DOTAP andDOTMAcationic polymers such as PEI and EPEI; peptides such as Xaa XaaXaa Lys Lys Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp TrpXaa Xaa Xaa, Lys Xaa Xaa Trp Trp Glu Thr Trp Trp Xaa Xaa Ser Gln Pro LysLys Xaa Arg Lys Xaa, Tyr Gly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp TrpGlu Thr Trp Trp Thr Glu Xaa, wherein any Xaa can be any amino acid.

The present invention can be better understood by reference to thefollowing actual examples demonstrating the operational capability ofthe invention. However. these examples are but one embodiment of thepresent invention, presented for the purpose of exemplifying theinvention in accordance with the requirements of the Patent Statute.These examples therefore do not represent the full scope of theinvention; reference is made to the claims that are appended hereto fora determination of the scope of the present invention.

EXAMPLE 1 Development of the Mouse Sepsis Model

E. coli SM101, a temperature-sensitive UDP-N-acetylglucosamineacyltransferase mutant that lose all detectable acyltransferaseactivity, and its wild-type K12, were i.p. injected as described belowto induce sepsis in mouse. SM101 has a defect in lipid A biosynthesisthat causes the outer membrane to be permeable to high-molecular-weightsubstances. The lipid A content of SM101 is reduced 2-3-fold comparedwith the wild-type. To prepare the bacteria for mouse infection, SM101were grown in LB medium at 37° C. Log-phase cultures of SM101 were grownto an optical density at 600 nm of 1.1 (equivalent to 5×108 CFU/ml),followed by centrifugation and resuspension in sterilephosphate-buffered saline (PBS) at 4° C. This log-phase preparation ofbacteria was serially diluted in PBS, and 3×10e8 CFU of bacteria wasi.p. injected to induce mouse sepsis. Serum was gathered andpro-inflammatory cytokines (IL-6, TNF, IL-1) and bacterial load tested,and mouse behavior monitored at various time points after injection. Allmice bled at every 24 hours. At 6 hours, mice showed evidence ofinfection (lethargy, warm to the touch, scruffy). As shown in FIG. 1,around 60% mice died within 48 hours after infection. As shown in FIG.2, 3 of 5 mice showed significant decrease in serum IL-6 concentration.Table 1 shows mouse bacterial load in blood after infection. Serumsample was collected at 24 hrs after infection, for cell growth assay bymeasuring viable cell count. Viable cell count was done by diluting thecultures and plating them on LB plates. The plates were then incubatedovernight at 37° C. and the number of colonies was enumerated by visualinspection.

EXAMPLE 2 Inhibition of Bacterial Growth by ODN

The inhibition of bacterial growth by ODN was evaluated by examining theeffect of DNA dose on the ability of ODN to inhibit SM101 growth. Inthis study, an ODN having the sequence CTC ATA CTC T was added to the1/50 diluted O/N SM101 cell cultures, at final concentration of 40 μM or400 μM, with addition of equal volume water as a negative control, andincubated with shaking at 30° C. After 2, 4 or 6 h, the growth wasmeasured by either the optical density at 600 nm (OD600) or viable cellcount, which was done by diluting the cultures and plating them intriplicate on LB plates with streptomycin. As shown in FIG. 3, uponaddition of ODN, cell growth was inhibited by 86-96%.

EXAMPLE 3 Inhibition of Bacterial Growth by ODN Expression Plasmid

In this study, the ODN expression plasmid As080103, having the sequenceCYGXO80103 listed above, and plasmid pssxGb without ODN insert asnegative control, were transformed into E. coli XL10-gold(kan). Theresulting cell cultures were plated on LB media with chloramphenicol andincubated at 37° C. O/N. As shown in FIG. 4, no XL10-gold(kan) carryingODN expression plasmid grew on the LB media.

EXAMPLE 4 Establishing Lethal Dose (LD70 ) in the Mouse Model

Six-week-old mice Balb/c (in groups of five) were used for infectionexperiments. A serial dilution of SM101 was injected intraperitoneally(i.p.) into mice in 400-μl aliquots. The animals were observed for 100h. Mice inoculated with bacteria were scored for their state of healthon a scale of 5 to 0, based on progressive disease states reflected byseveral clinical signs. A normal and unremarkable condition was scoredas 5; slight illness, defined as lethargy and ruffled fur, was scored as4; moderate illness, defined as severe lethargy, ruffled fur, andhunched back, was scored as 3; severe illness, with the above signs plusexudative accumulation around partially closed eyes, was scored as 2; amoribund state was scored as 1; and death was scored as 0. While theexperiments were not conducted in a double-blind manner, all animalswere evaluated by two or more independent observers. The signs of sepsiswill be also detected by relative change in both cytokines/chemokinesand clearance/persistence of organisms from peritoneal cavity andspleen. These experiments established that 109 CFU of strain SM101 isthe LD70 for 6-week-old mice Balb/c, with ˜60% mice that received theLD70 dose dying within 48 h. This LD70 was used in all the DNA therapyexperiments described in this study. A similar approach was used toestablish the LD70 for E.coli strain K-12.

EXAMPLE 5 Treatment of Sepsis using ODN

The efficacy of ODN therapy was evaluated in two separate experimentsusing the above-described SM101 bacteremia mouse model. The firstexamined the effect of DNA dose on the ability of ODN to rescue micefrom SM101 bacteremia. The second studied the effect on the outcome ofdelaying treatment for various periods. In the dose-ranging study, fivegroups of mice (five mice in each) were challenged by i.p. injection ofthe LD70 of SM101. Each of these groups was treated with a singleinjection of the ODN CTC ATA CTC T, administered i.p. immediately afterthe bacterial challenge at 4 nmol, 40 nmol, 400 nmol and 0 nmol. As anadditional control, a fifth group (two mice) was not challenged withbacteria, receiving only the injection of ODN (at the highest dose). Thestate of the health of these animals was monitored for one week. FIG. 1shows survival of infected mice with or without ODN treatment.

EXAMPLE 6 Delivering Plasmid to Target Bacterial Cells by BacteriophageT3 Extracts

A standard DNA packaging reaction mixture (25 μl) contains 0.5 mg thesaid plasmid DNA, 2×1010 phage equivalent(peg) of prohead, 20 pmol of gp18 and 3 pmol of gp 19 in complete pac buffer. The reaction mixture wasincubated at 30° C. for 30 min for DNA packaging and the reaction wasterminated by the addition of 1 μl of 2 mg/ml of DNase I. Afterincubation at 30° C. for 20 min, the filled heads are converted toinfectious particles by incubation with a head acceptor extractcontaining tail and tail fiber proteins. Proheads are prepared through asucrose gradient centrifuge of lysates of bacterial cells infected withbacteriophage T3, as described by Nakasu et al. (Nakasu et al., 1983,Virology, 127, 124-133). gp 18 and gp 19 proteins are purified asdecribed by Hamada et al (Hamada et al., 1986, Virology, 151, 110-118).The head acceptor extracts are isolated from the lysates of bacterialcells infected by bacteriophage T3, and purified through ammoniumsulfate precipitation. The resulting infectious particles contain thesaid ODN expression plasmid and are used to deliver the ODN expressionplasmid to the target bacterial cells.

Although described with reference to the figures and specific examplesset out herein, those skilled in the art will recognize that certainchanges can be made to the specific elements of the invention that aredescribed herein without changing the manner in which those elementsfunction to achieve their intended results. All such changes andmodifications which do not depart from the spirit of the presentinvention are intended to fall within the scope of the followingnon-limiting claims. TABLE 1 Mouse bacterial load after infectionSepticemia: Mice Bled at 24 hours Post Infection # Mice Bled CFU/ml*Control 6 0, 0, 3.6 × 10³, 1.6 × 10³, 4.6 × 10³, >10⁵ PNA [10 uM] 4 0,0, 0, 0 IP, PNA [100 uM] 4 0, 0, 0, 0*Septicemic mice succumbed to infection prior to 46 h.

1. The ODN expression plasmid As080103.
 2. A method of treatment ofsepsis in an animal patient comprising the steps of contacting thecausative agent of sepsis with an ODN comprising a sequence targeted toa specific gene of the causative agent for altering the expression ofthe specific gene to inhibit growth of the causative agent, kill thecausative agent, or inhibit the synthesis or secretion of toxin by thecausative agent.
 3. A DNA, RNA, or PNA oligonucleotide for treatment ofsepsis having one or more of the following nucleotide sequences: 5′(CTTTCA ACA GTT TTG ATG ACC TTT (SEQ. ID No. 1) GCT GAC CAT ACA ATT GCG ATATCG TGG GGA GTG AGA G)3′; 5′(CTC ATA CTC T)3′; (SEQ. ID No. 2) or 5′(GTTTCG AAG GCT AGC TAC AAC GAT (SEQ. ID No. 3) CAT CCA G)3′.


4. A cell having one or more of the sequences of claim 4 transformedtherein.